absence of heterosis in hybrid crested newtsclades a and b are distantly related with an estimated...

Absence of heterosis in hybrid crestednewtsJan W. Arntzen1, Nazan Üzüm2, Maja D. Ajdukovi�c3, Ana Ivanovi�c1,4

and Ben Wielstra1,5,6

1 Naturalis Biodiversity Center, Leiden, The Netherlands2 Department of Biology, Faculty of Arts and Sciences, Adnan Menderes University,Aydin, Turkey

3 Institute for Biological Research “Siniša Stankovi�c”, University of Belgrade, Belgrade, Serbia4 Institute of Zoology, Faculty of Biology, University of Belgrade, Belgrade, Serbia5 Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK6 Department of Ecology and Evolutionary Biology, University of California, Los Angeles,CA, USA

ABSTRACTRelationships between phylogenetic relatedness, hybrid zone spatial structure, theamount of interspecific gene flow and population demography were investigated,with the newt genus Triturus as a model system. In earlier work, a bimodal hybridzone of two distantly related species combined low interspecific gene flow withhybrid sterility and heterosis was documented. Apart from that, a suite of unimodalhybrid zones in closely related Triturus showed more or less extensive introgressivehybridization with no evidence for heterosis. We here report on populationdemography and interspecific gene flow in two Triturus species (T. macedonicus andT. ivanbureschi in Serbia). These are two that are moderately related, engage in aheterogeneous uni-/bimodal hybrid zone and hence represent an intermediatesituation. This study used 13 diagnostic nuclear genetic markers in a population atthe species contact zone. This showed that all individuals were hybrids, with noparentals detected. Age, size and longevity and the estimated growth curves are notexceeding that of the parental species, so that we conclude the absence of heterosisin T. macedonicus–T. ivanbureschi. Observations across the genus support thehypothesis that fertile hybrids allocate resources to reproduction and infertile hybridsallocate resources to growth. Several Triturus species hybrid zones not yet studiedallow the testing of this hypothesis.

Subjects Biogeography, Evolutionary Studies, Genetics, ZoologyKeywords Contact zone, Age distribution, Serbia, Hybrid zone, SNP markers, Skeletochronology

INTRODUCTIONHybrid zones are regions where genetically distinct populations meet and hybridize(Barton & Hewitt, 1985; Harrison, 1993). They provide natural settings for the study ofspeciation. In particular they allow research on the consequences of new, previouslyuntested genetic combinations of differentiated genomes in nature. Hybrid zones may takeseveral forms, from long and narrow zones to large areas of overlap and mosaics(Arnold, 1992). The positive relationship between the degree to which hybridizationproceeds and the phylogenetic relatedness of the species involved is well established

How to cite this article Arntzen et al. (2018), Absence of heterosis in hybrid crested newts. PeerJ 6:e5317; DOI 10.7717/peerj.5317

Submitted 15 May 2018Accepted 4 July 2018Published 24 July 2018

Corresponding authorJan W. Arntzen,[email protected]

Academic editorJohn Measey

Additional Information andDeclarations can be found onpage 12

DOI 10.7717/peerj.5317

Copyright2018 Arntzen et al.

Distributed underCreative Commons CC-BY 4.0

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(Jiggins & Mallet, 2000). In spite of some variation in the strength of reproductive isolationfor individuals from spatially isolated populations, or among different species pairs fromthe same evolutionary lineages (Seehausen et al., 2014; Kearns et al., 2018) accumulatedevidence suggests that, in diploid animals, reproductive isolation increases andintrogression between lineages decreases with divergence (Singhal & Moritz, 2013;Arntzen, Wielstra & Wallis, 2014; Beysard & Heckel, 2014; Dufresnes et al., 2014;Montanari et al., 2014; Taylor et al., 2014). Unfortunately, much less is known about howspecies’ ecological preferences—and with that species distributions and local spatialconfigurations—may affect hybridization and vice versa, and how phylogenetic relatednessaffects the interaction with ecology. Yet, such knowledge is relevant for the understandingof hybrid zones and the evolutionary inferences we draw from them. As Barton & Hewitt(1985: 121) proposed ‘ : : : as soon as the loss of fitness through hybridization inside thezone becomes small enough : : : the zone will collapse into broad sympatry’. Accordingly,the more closely related hybridizing species are, the more hybridization will take place,over yet smaller areas. A negative relationship between the degree of hybridization and theamount range overlap was indeed noted by Zuiderwijk (1980) in a variety of Europeanamphibian species pairs, but geographical variation and ecological differences remain to bestudied.

We propose that a good group to investigate if reproductive isolation accumulatesgradually would fulfil the following requirements. It would (i) be monophyletic and(ii) show more or less contiguous species ranges where (iii) closely as well as distantlyrelated species engage in hybrid zones and (iv) ecological profiles of the species aredifferent. Finally, (v) to reduce the impact of spatial scale a low dispersal capability wouldbe an asset. One group that qualifies is the European newt genus Triturus. The genus isdiverse and has a pan-European distribution, with outer ranges up to the Atlantic andthe Mediterranean and reaching into Scandinavia, Russia, the Caucasus and Iran.The species group inner ranges form a patchwork and all nine species are, in one placeor the other, involved in intra-generic hybridization (Arntzen, Wielstra & Wallis, 2014;Wielstra et al., 2014b). The genus Triturus is composed of three clades, namely the Triturusmarmoratus group (or marbled newts) with two species (clade A), the T. cristatusgroup with four species (clade B) and the T. karelinii group with three species (clade C).Clades A and B engage in western Europe and clades B and C meet up in southeasternEurope (Arntzen, 2003; Wielstra et al., 2014b). See Fig. 1 for species distributions andphylogenetic relationships.

Clades A and B are distantly related with an estimated 27.6 Ma period of lineageindependence (Wielstra & Arntzen, 2011). At their hybrid zone in France, T. marmoratus(clade A) and T. cristatus (clade B) interspecies F1 hybrids are infrequent (ca. 4% ofthe total population) and introgression is rare (

well-understood ecological differentiation. Forested and hilly areas are mostly occupiedby T. marmoratus and open and flat areas have T. cristatus (Schoorl & Zuiderwijk, 1981;Visser et al., 2017). Representatives of clades B and C—together known as crestednewts—show an intermediate level of phylogenetic relatedness with 10.4 Ma of lineageindependence. European species involved in the contact are T. cristatus, T. dobrogicus andT. macedonicus in clade B and T. ivanbureschi in clade C. Among these four, T. dobrogicusstands out as a lowland species (Vörös et al., 2016). The species meet up in the Balkanpeninsula along the lower Danube and in a wide zone running from Belgrade (Serbia) toThessaloniki (Greece) (Fig. 1). Species within the B and C clades have 8.8–5.3 Ma of lineageindependence and engage in a variety of more or less unimodal hybrid zones, i.e. with amajority of hybrids and few or no parentals (Arntzen, Wielstra & Wallis, 2014; Wielstraet al., 2017a, 2017b), with no evidence for heterosis so far. For data reviews in Triturussee Arntzen (2000) and Lukanov & Tzankov (2016). Because of the intermediate level ofrelatedness, the genetic and spatial interactions among species in the B and C clades are ofspecial interest. We here studied hybridization between T. macedonicus and T. ivanbureschiwhere they meet in southeastern Serbia. A phenotypically mixed population wasexamined with a panel of nuclear genetic markers to estimate ancestry and heterozygosityand individual age was estimated by skeletochronology. We searched for heterosis byanalysing hybrids for longevity, body size and growth.

Figure 1 Distribution of nine Triturus species over Europe and the Near East. Distribution of nineTriturus species over Europe and the Near East (afterWielstra & Arntzen, 2011). Major clades are (A) themarbled newts, (B) the T. cristatus species group of crested newts and (C) the T. karelinii species group ofcrested newts. Note that the spatial contact of clades is limited to central France (clades A and B) and thesouthern Balkans (B and C). Heterosis in size and longevity was observed in A � B hybrids (Mayenne,France, white dot, Francillon-Vieillot, Arntzen & Géraudie, 1990) and not in B� C hybrids (Vlasi, Serbia,asterisk, present paper). The insert shows the species phylogeny.

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Figure 2 Von Bertalanffy growth curves for Triturus newts. (A) Triturus cristatus (orange),T. marmoratus (green) and Triturus cristatus—T. marmoratus F1 hybrids (black) from Mayenne, France.Data are from Francillon-Vieillot, Arntzen & Géraudie, (1990). Females are larger than males of the sameage. The hybrids become larger and older than the parental species. No model could be fitted forT. marmoratus females. (B) Triturus macedonicus � T. ivanbureschi hybrid population from Vlasi insoutheastern Serbia (black lines), T. macedonicus from Montenegro (pink) and T. ivanbureschi popu-lations (blue) at localities Keşan and Klaros. Reference data are from Cvetkovi�c et al. (1996), Üzüm (2006),Üzüm & Olgun (2009) and Supplemental Information S6). Females are larger than males of the same agein three out of four populations. The Vlasi populations of hybrids shows no evidence for hybrid vigour insize, growth or longevity. The parameters that describe the Von Bertalanffy growth curves and theconfidence intervals are presented in Supplemental Information S5.



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MATERIALS AND METHODSPost-metamorphic crested newts were caught with funnel traps from March to July2013 in a pond near the village Vlasi in the southeast of Serbia (43.00 N, 22.64 E, altitude468 m a.s.l.). The focal newt population we henceforth refer to as ‘Vlasi.’ In total, 336individuals were measured and marked and a small part of the newt’s tail at its tip wastaken for genetic analyses (Arntzen, Smithson & Oldham, 1999). The population samplewas classified in five groups determined from external morphology and colourationcharacteristics with documented phenotypes as a starting point (Wallis & Arntzen, 1989;Arntzen & Wallis, 1999; Arntzen, 2003; Wielstra et al., 2013b). Classes were as follows:group 1—T. macedonicus-like (N = 26), group 2—leaning towards T. macedonicus(N = 48), group 3—intermediate phenotype (N = 146), group 4—a phenotype leaningtowards T. ivanbureschi (group 4, N = 83) and group 5—T. ivanbureschi-like (N = 33).Permission for fieldwork, for marking and to collect tissue samples was obtained fromthe Ministry of Energy, Development and Environmental Protection of the Republic ofSerbia (permit no. 353-01-35/2013-08).

Molecular data were gathered for a panel of nuclear encoded SNP markers (see below).We included reference samples with N = 3 for seven populations of T. ivanbureschi andseven populations of T. macedonicus available from Wielstra et al. (2013a) (Table 1).Reference populations were located to the south of the Vlasi population, distant fromother, more northerly distributed Triturus species. Wielstra et al. (2014a) producedsequence data for 52 short (ca. 140 bp) nuclear markers positioned in 3′UTR regions ofprotein-coding genes for three individuals from four populations positioned throughoutthe ranges of both T. ivanbureschi and T. macedonicus. We focussed on the subset of24 nuclear markers with species diagnostic allele variants for T. ivanbureschi andT. macedonicus. Additionally an mtDNA SNP (nd4) was designed from Sanger sequencedata taken from Wielstra et al. (2013a). We determined diagnostic SNPs by checking thesequence alignments by eye in MacClade 4.08 (Maddison & Maddison, 2005).

Genotyping was conducted commercially at the SNP genotyping facility of the Instituteof Biology, Leiden University, using the Kompetitive Allele-Specific PCR (KASP)genotyping system (LGC KBioscience, Teddington, UK). This involves fluorescence-basedgenotyping using SNP-specific primers. We used the program Kraken to design twoforward or reverse primers that are specific for (i.e. with a final base complementary to)one of the two potential SNP variants, in addition to a general reverse or forward primer(Semagn et al., 2014). We genotyped 378 crested newts in total: 21 individuals of bothparental species (the eight populations on which SNP identification was based plus anadditional six populations) and 336 individuals from Vlasi. Sequence alignments andKraken input for all markers are available in Supplemental Information S1. Raw output ofthe KASP genotyping protocol is in Supplemental Information S2. The Ion Torrent next-generation sequence data of Wielstra et al. (2014a) used for SNP discovery are availablefrom Dryad Digital Repository entry DOI 10.5061/dryad.36775. The geneticand morphological data used in the analysis are presented in SupplementalInformations S3 and S4.


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The program NewHybrids (Anderson & Thompson, 2002) was used to estimate theproportion of the Vlasi population consisting of recently formed hybrids. Referencepopulations were invoked with the ‘z-option’, otherwise settings were default.The program HIest was used to estimate heterozygosity (H) and ancestry (S)(Fitzpatrick, 2012). To evaluate the consistency and independence of the genetic dataand to assess hybrid zone structure Hardy–Weinberg equilibrium (HW) and linkagedisequilibrium (LD) were quantified with GenePop (Raymond & Rousset, 1995).

All animals encountered were marked by clipping the middle toe on the right foot forthe purpose of a population size estimate by the capture-recapture method, with resultsnot here reported. In a subsample of individuals (N = 170, 77 males, 85 females and eightsmall individuals with secondary sexual characters not expressed, i.e. juveniles) thatrepresented the five morphological groups, a phalanx of the toe was taken for agedetermination by skeletochronology. Lines of arrested growth (LAGs) were counted as inFrancillon-Vieillot, Arntzen & Géraudie (1990). Endosteal resorption was estimated bycomparing the diameters of eroded marrow cavities of adults with the diameters ofnon-eroded marrow cavities of juveniles and was observed in 75 males (97.4%), 72 females(91.7%) and two juveniles. Individual ages were estimated taking endosteal resorptioninto account. We also determined age at maturity from ‘rapprochement’. This is thetightening of LAGs that is associated with the shift of resources from growth to

Table 1 Populations of Triturus macedonicus, T. ivanbureschi and the mixed focal population fromVlasi, Serbia, genotyped with a panel of nuclear genetic markers.

Taxon, locality, country Coordinates Populationused formarker design

Samplesize

Tissue collectionnumber

Latitude Longitude

Triturus macedonicus

Paliambela, Greece 38.909 20.970 3 2815-7

Kerameia, Greece 39.562 22.081 Yes 3 3775-7

Kounoupena, Greece 39.683 19.764 Yes 3 2820-2

Lushnjë, Albania 41.000 19.664 3 3472-4

Vrbjani, Macedonia 41.413 20.816 3 3327, 3583-4

Gostivar, Macedonia 41.817 20.899 Yes 3 3601-3

Bjeloši, Montenegro 42.374 18.907 Yes 3 3245-7

Triturus ivanbureschi

Shumnatitsa, Bulgaria 42.297 23.626 3 2779-81

Saint Kosmas, Greece 41.084 24.669 3 2846-8

Alexandrovo, Bulgaria 42.601 25.093 Yes 3 2492-4

Keşan, Turkey 40.917 26.633 Yes 3 2360-2

Salihler, Turkey 39.181 26.826 3 1808-9, 1812

Alepu, Bulgaria 42.348 27.714 Yes 3 2602-4

Kocabey, Turkey 39.352 28.217 Yes 3 1879-81

Focal population

Vlasi, Serbia 42.998 22.637 336 6284-6620

Note:Tissus are kept at Naturalis Biodiversity Center (https://www.naturalis.nl/en/).


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reproduction. In some cases we observed a line formed at metamorphosis. Someexamples are in Fig. 3.

Body size was measured in millimetre from the tip of the snout up to the posterior endof the cloaca (snout vent length, SVl). Sexual dimorphism was estimated with the Lovich &Gibbons (1992) sexual dimorphism index SDi = (mean length of the larger sex/meanlength of the smaller sex) minus unity, and arbitrarily defined as positive when femalesare larger than males and negative in the reverse case. Von Bertalanffy growth curveswere determined with the function ‘growthmodels’ in the R package FSA (R Core Team,2013; FSA, 2017). Since the morphological groups that we distinguished showed nomarked genetic differentiation (see below) the data on size, age and growth were pooledfor both sexes. The few juveniles in the sample remained unsexed and were excludedfrom the analyses. Confidence intervals were estimated by bootstrapping with nlstools in

Figure 3 Cross-sections of a phalanges of Triturus macedonicus � T. ivanbureschi crested newt fromVlasi. Cross-sections of a phalange of a juvenile, male and female Triturus macedonicus � T. ivanbureschicrested newt from Vlasi. Study sites: m.c., marrow cavity; m.l., metamorphosis line; e.r., endostealresorption; p., periphery; rap., rapprochement. Note that the periphery is not regarded as a LAG. Sectionsare 18 mm thick and taken at the level of the diaphysis. (A) Three-year-old juvenile, SVl 48 mm. ThreeLAGs (arrow heads) are observed in the periosteal bone as well as a metamorphosis line (arrow). (B) Five-year-old male, SVl 64 mm. Four LAGs are observed in the periosteal bone (arrow heads). Endostealresorption present (arrow). (C) Six-year-old female, SVl 64 mm. Five LAGs are observed in the periostealbone (arrow heads). Endosteal resorption and rapprochement are also marked by arrows.



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2000 bootstrap iterations (Nlstools, 2015). Differences between morphological groupsin the level of estimated ancestry and heterozygosity were tested by ANOVA.The relationship between level of heterozygosity and SVl was tested by the Pearsoncorrelation coefficient. ANOVA and correlations were done with SAS software(SAS Institute Inc., 2011).

RESULTSOut of the candidate nuclear markers two (cnppd and kdm3) were dropped because adiagnostic SNP could not be identified and for four (ace, eif4ebp2, ssh2 and syncrip) theKraken software could not design a suitable set of primers. For the 18 remainingdiagnostic nuclear markers for which assays could be designed, PCR amplification forone (amot) failed. The locus slc25 yielded a high frequency of missing data (16%) in thereference populations, suggesting it did not amplify the T. ivanbureschi-allele well andresults for this marker were also discarded. For the remainder missing data amountedto 1.8%. The four loci ahe, ddx17, dnaj and sre showed highly significant LD for allsix pairwise combinations. This result was interpreted as tight physical linkage in a singlelinkage group. To increase data independence results for the locus ahe (with the fewestmissing data) were retained and the others dismissed. Subsequent tests for HW andLD yielded no significant comparisons under Bonferroni correction. The KASPgenotyping output for the remaining 13 markers is summarized in SupplementalInformation S3. One nuclear SNP marker (col18) showed a single instance ofheterozygosity in a parental individual, suggesting either a genotyping error or amarker that is not fully diagnostic. With N = 82 (21.7%) of missing data the mtDNAmarker (nd4) performed relatively poorly. All newts from the Vlasi population thatcould be genotyped possessed the mtDNA haplotype typical for T. ivanbureschi,as expected for this marker in this system (for background information seeWielstra et al., 2017a).

In NewHybrids none of the individuals from the Vlasi population were allocated toeither of the parental species, to the F1 hybrid class or to the class of backcrossestowards T. macedonicus. Several individuals were allocated to the ‘backcross toT. ivanbureshi class’ (N = 8, 2.4%). The majority of individuals was classified as F2 hybrids(N = 328, 97.6%). When NewHybrids was instructed to take not two but threegenerations into account, the majority (99.7%) was allocated to that third generation.HIest grouped all Vlasi individuals in the middle of the ancestry times heterozygositybivariate plot, somewhat off-centre in the direction of T. ivanbureschi (Fig. 4), with thereference populations in the lower left (pure T. macedonicus) and lower right corners(pure T. ivanbureschi). To explore possible differences between morphological groupsin the level of estimated ancestry (S) and heterozygosity (H) ANOVA was used overfive (groups 1–5) and three morphological groups (groups 1, 2–4 pooled, 5) with nostatistically significant results. Pearson correlation coefficients for S and SVl and H and SVlwere also not significant, for males as well as for females.

The body size distribution showed that females were significantly larger thanmales (SVl males 63.9, SVl females 67.5 mm; Student’s t-test P < 0.0001; SDi = 0.056).


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The age distribution for males and females showed no significant difference(Mann–Whitney U-test, P > 0.05; Table 2). In both sexes >70% of the individuals hadan estimated age of 5, 6 or 7 years. Age at maturity estimated by rapprochement wasmostly 2, 3 or 4 years in both sexes (average males 3.1, N = 75; average females 3.0,N = 74; Supplemental Information S4.). The absence of an observable rapprochementwas more frequent in females (12.9%) than in males (2.6%) (P < 0.05, G-test ofindependence), perhaps suggesting a less drastic shift in resource allocation in thetransition from growth to reproduction in females. Longevity was 13 years in males and11 years in females. The parameters that describe the Von Bertalanffy growth curvesand their confidence intervals are presented in Supplemental Information S5. Results forthe Vlasi populations were compared with data from the literature (Cvetkovi�c et al., 1996and Supplemental Information S6 on T. macedonicus and Üzüm, 2006 and Üzüm &Olgun, 2009 on T. ivanbureschi). In three out of four populations females are largerthan males of the same age. The other growth curves for populations and species aresimilar, with no more than 10 mm difference in SVl among the most differentgroups (Fig. 2B). Unlike T. cristatus � T. marmoratus hybrids, the T. macedonicus �T. ivanbureschi hybrids showed no heterosis. Longevity was higher in T. macedonicusthan in T. ivanbureschi, with interspecific hybrids from the Vlasi population takingan intermediate position.

Figure 4 Ancestry versus heterozygosity plot based on 13 species diagnostic nuclear genetic markers.Individuals from the Vlasi population are shown by open round symbols (N = 336). The left corner of thetriangle corresponds to Triturus macedonicus (solid square symbols, N = 21), the right corner toT. ivanbureschi (solid triangle symbol, N = 21) and the upper corner to (non-observed) F1 hybridspopulations. The Vlasi populations of hybrids shows no evidence for hybrid vigour in size, growth orlongevity. Full-size DOI: 10.7717/peerj.5317/fig-4


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DISCUSSIONWe encountered a population of crested newts near Vlasi in southeastern Serbia thatshowed extensive phenotypic variation and—being close to the territory of bothT. macedonicus and T. ivanbureschi—a hybrid nature was assumed. We were, however,unable to determine the extent of hybridization from morphological and colourationcharacters and yet wanted to find out if this hybrid zone population has a unimodalcharacter (with a majority of hybrids and few or no parentals), a bimodal character(with predominantly the parental species and few hybrids), or something in between.We were also interested in fitness consequences that hybridization might have on thedemography of the population, in particular if heterosis would be combined withhybrid sterility.

All individuals from the focal population were allocated to the second generationof hybrids, indicating strong admixture along with the absence of recent crossingsbetween the parental species. When a third generation was an option, individualswhere allocated to that third generation, further supporting the absence of recenthybrids. Looking yet deeper into the coalescence would require more genetic markers.An ancestry versus heterozygosity plot (Fig. 4) further support that genotypes in thisVlasi population result from many generations of admixture. In the absence of selectionand excluding physical linkage LD halves every next generation and will barely bemeasurable after a few generations (Barton & Gale, 1993). Accordingly, the absenceof admixture LD also suggests that hybridization between T. macedonicus andT. ivanbureschi has been ongoing for more than a couple of generations. We conclude

Table 2 Age distribution of juveniles, males and females in the admixed Triturus macedonicus–T.ivanbureschi population from Vlasi, Serbia.

Age Juveniles Males Females

0

1

2 2

3 5 3

4 1 6 5

5 18 16

6 20 24

7 18 20

8 9 11

9 4 3

10 1

11 3

12

13 1

Total 8 77 85

Note:Ages are estimated by skeletochronology.


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that Vlasi is a population with only hybrids and the parental species absent. A hybridizedpopulation was also suggested by the absence of genetically diagnosable morphologicalgroups. These results suggest that T. macedonicus and T. ivanbureschi engage in aunimodal hybrid zone. The genetic affiliation of the Vlasi population is somewhatcloser to T. ivanbureschi than to T. macedonicus (Fig. 4). This is in line with thedocumented distributions of the two species where the Vlasi population is geographicallycloser to T. ivanbureschi than to T. macedonicus (Wielstra et al., 2014b). However,the exact position of the centre and the width of the cline remain to be documented.This result contrasts with the situation to the northwest, where the two speciesengage over a wide area of species replacement. The mosaic distribution includes aT. ivanbureschi enclave that was cut off from its main distribution by supersedingT. macedonicus (Arntzen, 2003; Arntzen &Wallis, 1999, cf. Fig. 1). The sole transmissionof the mitochondrial haplotype typical for T. ivanbureschi, that we here confirmedfor the Vlasi hybrid population, helped to reconstruct a scenario in whichT. macedonicus advanced at the expense of T. ivanbureschi (Wielstra et al., 2017a).

Narrow zones with extensive hybridization are found at contacts between crestednewts species belonging to clades B and C (Arntzen, Wielstra & Wallis, 2014;Wielstra et al., 2017b, cf. Fig. 1). A relationship appears to exist with species relatedness,so that closely related species form clines and more distant species engage in amosaic distribution, as described for T. cristatus and T. marmoratus. The presenceof a unimodal (clinal) as well as bimodal (mosaic) hybrid zone structure in theT. macedonicus–T. ivanbureschi contact is in line with an intermediate level ofrelatedness.

CONCLUSIONAs demonstrated here, the T. macedonicus–T. ivanbureschi hybrids are fertile, producingnew genetic combinations that themselves reproduce in a more or less narrow hybrid zone.This result for a comparison of the B and C clades contrasts with the results obtainedearlier for T. cristatus and T. marmoratus in the A and B clades, where hybridizationin is an evolutionary dead-end. These hybrids are largely sterile and introgression isnear-absent (Arntzen & Wallis, 1991; Arntzen et al., 2009). We propose that in thegenus Triturus heterosis is restricted to hybrids that are infertile and that—also inadult stage—direct their resources more to growth than to reproduction. This is morelikely to be the case for genetically incompatible than for genetically compatible species.The testing grounds for this assertion are several unimodal hybrid zones of closely relatedTriturus species, i.e. within the A and B clades (Fig. 1) where demonstration ofheterosis would contradict our hypothesis.

ACKNOWLEDGEMENTSWe thank R. Bancila and F. Stanescu for help with curve fitting statistics, O. Schaapand K. Vrieling for running the SNP-line and three anonymous reviewers for helpfulcomments.


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ADDITIONAL INFORMATION AND DECLARATIONS

FundingB. Wielstra received funding from the European Union’s Horizon 2020 research andinnovation programme under the Marie Sk1odowska-Curie grant agreement No. 655487.M. D. Ajdukovi�c and A. Ivanovi�c were supported by Serbian Ministry of Education, Scienceand Technological Development grant no. 173043. A. Ivanovi�c was in receipt of a ‘Temminckgrant’ provided by Naturalis Biodiversity Center. The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscript.

Grant DisclosuresThe following grant information was disclosed by the authors:Marie Sk1odowska-Curie grant agreement No: 655487.Serbian Ministry of Education, Science and Technological Development grant no: 173043.Naturalis Biodiversity Center.

Competing InterestsThe authors declare that they have no competing interests.

Author Contributions� Jan W. Arntzen conceived and designed the experiments, analysed the data, contributedreagents/materials/analysis tools, prepared figures and/or tables, authored or revieweddrafts of the paper, approved the final draft.

� Nazan Üzüm performed the experiments, analysed the data, approved the final draft.� Maja D. Ajdukovi�c performed the experiments, approved the final draft.� Ana Ivanovi�c conceived and designed the experiments, analysed the data, contributedreagents/materials/analysis tools, authored or reviewed drafts of the paper, approved thefinal draft.

� Ben Wielstra conceived and designed the experiments, performed the experiments,analysed the data, contributed reagents/materials/analysis tools, authored or revieweddrafts of the paper, approved the final draft.

Animal EthicsThe following information was supplied relating to ethical approvals (i.e. approving bodyand any reference numbers):

The Ministry of Energy, Development and Environmental Protection of the Republic ofSerbia (today, Ministry of Environmental Protection, Agency for Nature Protection)provided full approval to the Institute of Biological Research “Siniša Stankovi�c” to conductresearch on the hybrid population (Vlasi) and for DNA tissue sampling. The permission isadministrated as No. 353-01-35/2013-08.

Data AvailabilityThe following information was supplied regarding data availability:

The raw data are provided in the Supplemental Files.


http://dx.doi.org/10.7717/peerj.5317#supplemental-informationhttp://dx.doi.org/10.7717/peerj.5317https://peerj.com/

Supplemental InformationSupplemental information for this article can be found online at http://dx.doi.org/10.7717/peerj.5317#supplemental-information.

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Absence of heterosis in hybrid crested newtsIntroductionMaterials and MethodsResultsDiscussionConclusionflink6References

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absence of heterosis in hybrid crested newtsclades a and b are distantly related with an estimated...

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